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Halobacillus halophilus (formerly Sporosarcina halophila) is an obligate, moderately halophilic bacterium which was isolated from salt marsh at the North Sea coast of Germany (1). Most interestingly, growth of vegetative cells as well as germination of endospores of H. halophilus is strictly chloride dependent (1a, 4). H. halophilus is the first obligate chloride-dependent bacterium known to date, and projects to study the physiological basis of the chloride dependence have just begun. In one of these projects, we noted that H. halophilus creates an electrochemical chloride potential (µ_Cl) of about 190 to 230 mV across the membrane (M. Roeßler and V. Müller, unpublished data). The µ_Cl is, in principal, sufficient to drive energy-dependent reactions. Flagellar rotation in bacteria is such an energy-dependent process, driven by the electrochemical ion gradient (Na⁺, H⁺) across the membrane (3). Since H. halophilus is motile by means of flagella, we asked whether µ_Cl is used as a driving force for flagellar rotation.

Motile cells were selected by several passages over swarm agar plates, which contained 2.96 g of NH₄NO₃, 1.21 g of Tris-HCl, 1 ml of vitamin solution (8), 2.1 mg of FeSO₄, 1 g of glucose, and 0.25 g of yeast extract per liter of artificial seawater (2). MgCl₂ was replaced by MgSO₄; NaCl and NaNO₃ were added separately in concentrations as indicated. The pH was adjusted to 7.5, and the incubation temperature was 30°C.

As can be seen in Fig. 1, H. halophilus was motile on swarm agar plates containing 1 M NaCl, as visible by the diffuse zone of bacteria around the colonies. It should be noted that H. halophilus is much more slowly moving than other bacteria, and, therefore, true chemotactic rings were not observed. To analyze the effect of Cl, NaCl was replaced by other sodium ion salts. It is evident from Fig. 1 that there is a specific requirement for the anion. Neither NaBr nor NaNO₃ or Na₂SO₄ could promote motility. Since at least NaBr and NaNO₃ have been shown to promote growth of H. halophilus in the absence of NaCl (4), the apparent nonmotility in the presence of NaNO₃ or NaBr is not due to a general inhibition of metabolism by these salts. These experiments demonstrate that motility of H. halophilus is strictly chloride dependent. To further characterize the chloride dependence, quantitative studies with swarm agar plates containing increasing Cl concentrations were performed. The salt concentration was always kept constant at 1 M by appropriate addition of NaNO₃. An increase in the Cl concentration led to a concomitant increase in motility of H. halophilus, as determined by the time-dependent increase of the swarm size (Fig. 2A). The optimum was reached at around 1.0 M Cl (Fig. 2B). This value is well within the range of the Cl concentration required for optimal growth, which was determined to be 0.5 to 2 M (4).

View larger version (93K): [in this window] [in a new window]   FIG. 1.   Chloride-dependent motility of H. halophilus. Swarm agar plates containing 1 M NaCl, 1 M NaBr, 0.66 M Na2SO4, or 1 NaNO3 were inoculated with H. halophilus and incubated for 48 h at 30°C.

View larger version (11K): [in this window] [in a new window]   FIG. 2.   Kinetic analyses of chloride-dependent motility of H. halophilus. Colonies were spotted on swarm agar plates with increasing NaCl concentrations. The salt concentration was kept constant at 1 M by appropriate addition of NaNO3. (A) The swarm sizes were measured at the time points indicated and plotted against the incubation time. (B) Migration rates were calculated and plotted against the chloride concentration.

To determine whether µ_Cl is the driving force for flagellar rotation, free-swimming cells suspended in complex medium (8 g of nutrient broth per liter, 50 mM MgSO₄, 1 M NaCl or NaNO₃) were observed under the phase-contrast light microscope. Cells grown overnight in chloride-containing medium were motile, whereas cells grown in chloride-free but nitrate-containing medium were not. Addition of chloride to NaNO₃-grown cultures did restore motility only after a further incubation for 10 to 12 h. These experiments are not in favor of Cl as the coupling ion for flagellar rotation.

Scanning electron microscopic investigations of cells grown in chloride- or nitrate-containing media were performed to analyze whether chloride might affect the synthesis of flagella. Therefore, cells were grown in complex medium supplemented with either 1 M NaCl or 1 M NaNO₃. Cells were harvested by centrifugation and fixed with 2.5% glutaraldehyde in fixative buffer essentially as described previously (7). In addition, the buffer contained 1 M NaCl or 1 M NaNO₃ to avoid effects caused by hypoosmosis. Samples were analyzed in a Hitachi S-4100 field emission scanning electron microscope, and representative electron micrographs are depicted in Fig. 3. As can be seen in Fig. 3A, cells grown in chloride-containing medium have one flagellum per cell. In cells grown in chloride-free, nitrate-containing medium, the cell surface had a rough appearance (Fig. 3B). In addition, fibrillar structures extruding into the medium were sometimes visible. These structures have a significantly smaller diameter than flagella and, therefore, most likely are not flagella which are deformed due to lack of chloride. In the presence of NaNO₃, cell size was increased and cells tended to aggregate. Most interestingly, we could never observe any flagella in cells grown in nitrate-containing media. A possible inhibition of flagellar synthesis by nitrate could be excluded since flagellation of cells grown in the presence of both 1 M NaCl and 1 M NaNO₃ was indistinguishable from cells grown in 1 M NaCl. (data not shown).

View larger version (109K): [in this window] [in a new window]   FIG. 3.   Chloride-dependent flagellar synthesis in H. halophilus. Cells were grown in complex medium supplemented with 1 M NaCl (A) or 1 M NaNO3 (B) and analyzed by scanning electron microscopy as described in the text.

This is the first demonstration of chloride-dependent flagellation and motility in any living cell. The finding of chloride-dependent synthesis of flagella adds another function to chloride in H. halophilus. The role of chloride could be to serve as an intracellular signal for transcription of flagellar genes or it could be involved in export and assembly of subunits and/or stability of the flagella. A molecular study addressing these questions has to await the generation of protein- and/or RNA-directed probes. However, since germination of endospores as well as growth of the moderate halophile H. halophilus is strictly chloride dependent, it is tempting to speculate that H. halophilus uses Cl as a signal to measure the salinity of its environment. Cl could trigger expression of genes, directly or indirectly, involved in flagellar synthesis. Cl-induced gene expression was recently observed in Lactococcus lactis (5, 6). The elucidation of the role of chloride in flagellar synthesis and assembly in H. halophilus is a challenging task for future experiments, which have just started in our laboratory.